Prenylation inhibitors and methods of their synthesis and use

ABSTRACT

The present invention is directed to compounds useful in the treatment of diseases associated with prenylation of proteins and pharmaceutically acceptable salts thereof, to pharmaceutical compositions comprising same, and to methods for inhibiting protein prenylation in an organism using the same.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. patent application Ser. No.11/237,134, filed Sep. 27, 2005, now U.S. Pat. No. 7,112,596, which is aDivisional of U.S. application Ser. No. 10/646,256, now U.S. Pat. No.6,960,603, filed Aug. 22, 2003, which is a Divisional of U.S.application Ser. No. 10/336,285, now U.S. Pat. No. 6,649,638, filed Jan.3, 2003, which is a Continuation-In-Part of U.S. patent application Ser.No. 10/219,628, filed Aug. 14, 2002, now abandoned. These prioritydocuments are incorporated herein, in their entirety, by this reference.

FIELD OF THE INVENTION

This present invention relates to a class of novel compounds useful inthe treatment of diseases associated with the prenylation of proteins.

BACKGROUND OF THE INVENTION

The mammalian Ras proteins are a family of guanosine triphosphate (GTP)binding and hydrolyzing proteins that regulate cell growth anddifferentiation. Their overproduction or mutation can lead touncontrolled cell growth, and has been implicated as a cause oraggravating factor in a variety of diseases including cancer,restenosis, psoriasis, endometriosis, atherosclerosis, viral or yeastinfection, and corneal neovascularization.

Ras proteins share characteristic C-terminal sequences termed the CAAXmotif, wherein C is Cys, A is an amino acid, usually an aliphatic aminoacid, and X is an aliphatic amino acid or other type of amino acid. Thebiological activity of the proteins is dependent upon thepost-translational modification of these sequences by isoprenoid lipids.For proteins having a C-terminal CAAX sequence, this modification, whichis called prenylation, occurs in at least three steps: the addition ofeither a 15 carbon (farnesyl) or 20 carbon (geranylgeranyl) isoprenoidto the Cys residue, the proteolytic cleavage of the last three aminoacids from the C-terminus, and the methylation of the new C-terminalcarboxylate. Zhang and Casey, Ann. Rev. Biochem. 1996, 65, 241-269. Theprenylation of some proteins may include a fourth step; thepalmitoylation of one or two Cys residues N-terminal to the farnesylatedCys.

Ras-like proteins terminating with XXCC or XCXC motifs can also beprenylated and are modified by geranylgeranylation on the Cys residues.These proteins do not require an endoproteolytic processing step. Whilesome mammalian cell proteins terminating in XCXC are carboxymethylated,it is not clear whether carboxymethylation follows prenylation ofproteins terminating with XXCC motifs. Clarke, Ann. Rev. Biochem., 1992,61, 355-386. For all Ras-like proteins, however, addition of theisoprenoid is the first step of prenylation, and is required for thesubsequent steps. Cox and Der, Critical Rev. Oncogenesis, 1992, 3,365-400; and Ashby et al., Curr. Opinion Lipidology, 1998, 9, 99-102.

Three enzymes have been found to catalyze protein prenylation:farnesyl-protein transferase (FPTase), geranylgeranyl-proteintransferase type I (GGPTase-I), and geranylgeranyl-protein transferasetype-II (GGPTase-II, also called Rab GGPTase). These enzymes are presentin both yeast and mammalian cells. Schafer and Rine, Annu. Rev. Genet.,1992, 30, 209-237. U.S. Pat. No. 5,578,477 discloses a method ofpurifying FPTase using recombinant technology and yeast host cells. Suchtechniques are useful in the elucidation of the enzyme structures.

FPTase and GGPTase-I are α/β heterodimeric enzymes that share a common αsubunit; the β subunits are distinct but share approximately 30% aminoacid identity. Brown and Goldstein, Nature, 1993, 366, 14-15; Zhang etal., J. Biol. Chem., 1994, 269, 3175-3180. GGPTase II has different αand β subunits, and complexes with a third component (REP, Rab EscortProtein) that presents the protein substrate to the α/β catalyticsubunits. GGPTase proteins, and the nucleic acid sequence encoding them,are disclosed by U.S. Pat. No. 5,789,558 and WO 95/20651. U.S. Pat. No.5,141,851 discloses the structure of a FPTase protein.

Each of these enzymes selectively uses farnesyl diphosphate orgeranylgeranyl diphosphate as the isoprenoid donor, and selectivelyrecognizes the protein substrate. FPTase farnesylates CAAX-containingproteins that end with Ser, Met, Cys, Gln or Ala. GGPTase-Igeranylgeranylates CAAX-containing proteins that end with Leu or Phe.For FPTase and GGPTase-I, CAAX tetrapeptides comprise the minimum regionrequired for interaction of the protein substrate with the enzyme.GGPTase-II modifies XXCC and XCXC proteins, but its interaction withprotein substrates is more complex, requiring protein sequences inaddition to the C-terminal amino acids for recognition. Enzymologicalcharacterization of FPTase, GGPTase-I and GGPTase-II has demonstratedthat it is possible to selectively inhibit only one of these enzymes.Moores et al., J. Biol. Chem., 1991, 266, 17438.

GGPTase-I transfers a geranylgeranyl group from the prenyl donorgeranylgeranyl diphosphate to the cysteine residue of substrate CAAXprotein. Clarke, Annu. Rev. Biochem., 1992, 61, 355-386; Newman andMagee, Biochim. Biophys. Acta, 1993, 1155, 79-96. Known targets ofGGPTase-I include the gamma subunits of brain heterotrimeric G proteinsand Ras-related small GTP-binding proteins such as RhoA, RhoB, RhoC,CDC42Hs, Rac1, Rac2, Rap1A and Rap1B. The proteins RhoA, RhoB, RhoC,Rac1, Rac2 and CDC42Hs have roles in the regulation of cell shape.Ridley and Hall, Cell, 1992, 70, 389-399; Ridley et al., Cell, 1992, 70,401-410; Bokoch and Der, FASEB J, 1993, 7, 750-759. Rac and Rap proteinsplay roles in neutrophil activation.

It has been found that the ability of Ras proteins to affect cell shapeis dependant upon Rho and Rac protein function. See, e.g., Mackey andHall, J. Biol. Chem., 1998, 273, 20688-20695. It thus follows thatbecause Rho and Rac proteins require geranylgeranylation for function,an inhibitor of GGPTase-I would block the functions of these proteins,and may be useful as, for example, an anticancer agent. This notion issupported by recently reported research.

For example, GGPTase-I inhibitors can arrest human tumor cells that lackp53 in G0/G1, and induce the accumulation of p21^(WAP). This suggeststhat these inhibitors could be used to restore growth arrest in cellslacking functional p53. Vogt et al., J. Biol. Chem., 1997, 272,27224-27229. Noteworthy in this regard are reports indicating thatK-Ras, the form of Ras gene most associated with human cancers, can bemodified by GGPTase-I in cells where FPTase is inhibited. Whyte et al.,J. Biol. Chem., 1997, 272, 14459-14464. Since geranylgeranylated Ras hasbeen reported to be as efficient as the farnesylated form in celltransformation studies, K-Ras cancers could be treated with GGPTase-Iinhibitors. Lerner et al., J. Biol. Chem., 1995, 270, 26770-26773.

In addition to cancer, there are other pathological conditions for whichGGPTase inhibitors may be used as intervention agents. These include,for example, the intimal hyperplasia associated with restenosis andatherosclerosis. Pulmonary artery smooth muscle cells seem particularlysensitive to inhibition of GGPTase-I, and treatment of such cells with aGGPTase inhibitor resulted in a superinduction of their induciblenitric-oxide synthase (NOS-2) by interleukin-1β. Finder et al., J. Biol.Chem., 1997, 272, 13484-13488.

GGPTase inhibitors may also be used as anti-fungal agents. In S.cerevisiae and Candida albicans, and apparently most other fungi, cellwall biosynthesis is controlled by a Rho-type protein that is modifiedby the fungal GGPTase-I. Qadota et al., Science, 1996, 272, 279-281.Selective inhibition of the fungal enzyme would diminish cell wallintegrity, and thus be lethal to fungal cells.

Numerous other prenylation inhibitors have been studied. Some examplesof these are disclosed by U.S. Pat. Nos. 5,420,245; 5,574,025;5,523,430; 5,602,098; 5,631,401; 5,705,686; 5,238,922; 5,470,832; and6,191,147; and by European Application Nos. 856,315 and 537,008. Theeffectiveness and specificity of these inhibitors vary widely, as dotheir chemical structures, and many of them are difficult to synthesizeand purify.

Therefore, there is a need for new, more effective prenyl-proteintransferase inhibitors.

SUMMARY OF THE INVENTION

One embodiment of the present invention provides compounds of thefollowing formula or pharmaceutically-acceptable salts thereof, andmethods of use thereof:

wherein

R₁ is a halogenated phenyl;

R₂ is nitrogen, phenyl, phenylamide, pyrazole, methylpyrazole,dimethylpyrazole, pyridine, thiophene, dimethylcyclobutyl,dimethylcyclopropyl or cyclopropyl;

R₃ is benzyl, isopropyl, chlorobenzyl, 2-benzyl-5-methyl-imidazole,methyl thiophene, trifluoro methyl benzyl, 3,5-trifluoromethyl benzyl,or ethylmethylsulfide; and,

R₄ is NH₂ or OH.

Another embodiment of the present invention provides the followingcompounds or pharmaceutically-acceptable salts thereof, and methods ofuse thereof:

wherein

R₁ is a halogenated phenyl;

R₂ is bicyclo[2.2.1]heptane (norbornane), cyclopropane or cyclohexane;and,

R₃ is NH₂, OH or 2-amino-3-phenylpropanamide (i.e. a compound of thestructure —NHCH(CH₂—C₆H₅)—CONH₂).

Another embodiment of the present invention provides the followingcompounds or a pharmaceutically-acceptable salts thereof, and methods ofuse thereof:

wherein

R₁ is a halogenated phenyl;

R₂ is (CH₂)_(X) where x is an integer between 1-4, cyclobutane ordimethylcyclobutane; and,

R₃ is NH₂ or OH.

Another embodiment of the present invention provides any of thefollowing compounds or pharmaceutically-acceptable salts thereof, andmethods of use thereof:

wherein R is a halogenated phenyl.

The present invention also provides a composition comprising a compoundof the present invention, or a pharmaceutically-acceptable salt thereof,and a pharmaceutically-acceptable carrier.

The present invention also provides a method for inhibiting proteinprenylation comprising contacting an isoprenoid transferase with acompound of the present invention or a pharmaceutically-acceptable saltthereof. As used herein, an “isoprenoid transferase” refers to anyenzyme capable of transferring an isoprenoid group, for example,farnesyl or geranylgeranyl, to a protein, e.g., Ras or Ras-likeproteins. Such isoprenoid transferases include FPTase, GGPTase I andGGPTase II. Unless the context requires otherwise, the term “contacting”refers to providing conditions to bring the compound into proximity toan isoprenoid transferase to allow for inhibition of activity of theisoprenoid transferase. For example, contacting a compound of thepresent invention with an isoprenoid transferase can be accomplished byadministering the compound to an organism, or by isolating cells, e.g.,cells in bone marrow, and admixing the cells with the compound underconditions sufficient for the compound to diffuse into or be activelytaken up by the cells, in vitro or ex vivo, into the cell interior. Whenex vivo administration of the compound is used, for example, in treatingleukemia, the treated cells can then be reinfused into the organism fromwhich they were taken.

Such method for inhibiting protein prenylation can be used, for example,in prevention and/or treatment of a disease or condition in a plant oranimal that is caused, aggravated or prolonged by Ras or Ras-likeprotein prenylation. In animals, such diseases include, but are notlimited to, cancer, restenosis, psoriasis, endometriosis,atherosclerosis, ischemia, myocardial ischemic disorders such asmyocardial infarction, high serum cholesterol levels, viral infection,fungal infections, yeast infections, bacteria and protozoa infections,and disorders related to abnormal angiogenesis including, but notlimited to, corneal neovascularization. In plants, such diseases includeyeast and viral infections.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “organism” includes plants and animals.Exemplary animals include mammals, fish, birds, insects, and arachnids.Humans can be treated with the compounds of the invention and fallwithin the mammal sub-category.

As used herein, the term “CAAX” means a C-terminal peptide sequencewherein C is Cys, A is an amino acid, usually an aliphatic amino acid,and X is another amino acid, usually Leu or Phe.

As used herein, the term “CAAX protein” means a protein comprising aCAAX sequence.

As used herein, the term “XXCC” means a C-terminal peptide sequencewherein C is Cys and X is another amino acid, usually Leu or Phe.

As used herein, the term “XXCC protein” means a protein comprising aXXCC sequence.

As used herein, the term “XCXC” means a C-terminal peptide sequencewherein C is Cys and X is another amino acid, usually Leu or Phe.

As used herein, the term “XCXC protein” means a protein comprising aXCXC sequence.

As used herein, the term “Ras or Ras-like protein” encompasses Rasproteins, brain heterotrimeric G proteins, and other GTP-bindingproteins such as members of the Rho, Rac and Rab family including, butnot limited to, RhoA, RhoB, RhoC, CDC42Hs, Rac1, Rac2, Rap1A and Rap1B.A Ras or Ras-like protein may be a CAAX, XXCC, or XCXC protein. The term“Ras or Ras-like protein” as used herein also encompasses Rheb,inositol-1,4,5,triphosphate-5-phosphatase, and cyclic nucleotidephosphodiesterase and isoforms thereof, including nuclear lamin A and B,fungal mating factors, and several proteins in visual signaltransduction.

As used herein, the term “Ras or Ras-like protein prenylation” means theprenylation of a Ras or Ras-like protein that is catalyzed or caused byGGPTase I, GGPTase II, or FPTase.

As used herein, the term “prenylation inhibitor” means a compound ormixture of compounds that inhibits, restrains, retards, blocks orotherwise affects protein prenylation, preferably Ras or Ras-likeprotein prenylation. A prenylation inhibitor may inhibit, restrain,retard, or otherwise affect the activity of GGPTase I, GGPTase II,and/or FPTase.

As used herein, the term “a pharmaceutically-acceptable salt thereof”refers to salts prepared from pharmaceutically-acceptable nontoxic acidsor bases including inorganic acids and bases and organic acids andbases. Examples of such inorganic acids are hydrochloric, hydrobromic,hydriodic, sulfuric, and phosphoric. Appropriate organic acids may beselected, for example, from aliphatic, aromatic, carboxylic and sulfonicclasses of organic acids, examples of which are formic, acetic,propionic, succinic, glycolic, glucuronic, maleic, furoic, glutamic,benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic,stearic, sulfanilic, algenic, tartaric, citric and galacturonic.Examples of suitable inorganic bases include metallic salts made fromaluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.Appropriate organic bases may be selected, for example, fromN,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumaine (N-methylglucamine), lysine and procaine.Preferred salts of the compounds of this invention are TFA and acetatesalts.

The phrase “therapeutically effective amount of prenylation inhibitor”as used herein means that amount of prenylation inhibitor which alone orin combination with other drugs provides a therapeutic benefit in thetreatment, management, or prevention of conditions in a plant or animalthat are caused, aggravated or prolonged by Ras or Ras-like proteinprenylation. Such conditions include, but are not limited to, cancer,restenosis, psoriasis, endometriosis, atherosclerosis, ischemia,myocardial ischemic disorders such as myocardial infarction, high serumcholesterol levels, viral infection, fungal infections, yeastinfections, bacteria and protozoa infections, and undesiredangiogenesis, abnormal angiogenesis or abnormal proliferation such as,but not limited to, corneal neovascularization. Other conditions includeabnormal bone resorption and conditions related thereto.

“Alkyl” groups according to the present invention are aliphatichydrocarbons which can be straight, branched or cyclic. Alkyl groupsoptionally can be substituted with one or more substituents, such as ahalogen, alkenyl, alkynyl, aryl, hydroxy, amino, thio, alkoxy, carboxy,oxo or cycloalkyl. There may be optionally inserted along the alkylgroup one or more oxygen, sulfur or substituted or unsubstitutednitrogen atoms. Exemplary alkyl groups include methyl, ethyl, propyl,i-propyl, n-butyl, t-butyl, bicycloheptane (norbornane), cyclobutane,dimethyl-cyclobutane, cyclopentane, cyclohexane, fluoromethyl,difluoromethyl, trifluoromethyl, chloromethyl, trichloromethyl, andpentafluoroethyl. Preferably, alkyl groups have from about 1 to about 20carbon atoms chains, more preferably from about 1 to about 10 carbonatoms, still more preferably from about 1 to about 6 carbon atoms, andmost preferably from about 1 to about 4 carbon atoms.

“Aryl” groups are monocyclic or bicyclic carbocyclic or heterocyclicaromatic ring moieties. Aryl groups can be substituted with one or moresubstituents, such as a halogen, alkenyl, alkyl, alkynyl, hydroxy,amino, thio, alkoxy or cycloalkyl.

“Heteroaryl” refers to monocyclic or bicyclic aromatic ring having atleast one heteroatom selected from nitrogen, sulfur, phosphorus andoxygen. Preferred heteroaryls are 5- and 6-membered aromatic ring whichcontain from about 1 to about 3 heteroatoms. Examples of heteroarylgroups include, but are not limited to, pyridinyl, imidazolyl,pyrimidinyl, pyrazolyl, (1,2,3)- and (1,2,4)-triazolyl, pyrazinyl,tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrrolyl, thiazolyl,pyrrole, thiophenyl, furanyl, pyridazinyl, isothiazolyl, andS-triazinyl.

“N-heteroaryl” refers to monocyclic or bicyclic aromatic ring having atleast one nitrogen atom in the aromatic ring moiety. ExemplaryN-heteroaryls include, but are not limited to, pyridinyl, imidazolyl,pyrimidinyl, pyrazolyl, (1,2,3)- and (1,2,4)-triazolyl, pyrazinyl,tetrazolyl, isoxazolyl, oxazolyl, pyrrolyl, pyrrole, pyridazinyl, andisothiazolyl. Preferably, N-heteroaryl is pyridinyl. More preferably,N-heteroaryl is pyridin-3-yl.

The term “aryl containing at least one nitrogen substituent” refers toan aryl moiety having a substituent such as an amino, including mono-,di-, and tri-alkyl amino groups; amido; or C₁-C₄ alkyl groups having anamino or an amido substituent. Preferably, an “aryl containing at leastone nitrogen substituent” is an aryl moiety having amino, amido or C₁-C₂alkyl having an amino or amido substituent; more preferably amino, amidoor C₁ alkyl having an amino or amido substituent; still more preferablyan amino or amido substituent; and most preferably an amino substituent.

The term “peptoids” or “polypeptoids” refers to poly-(N-substitutedglycine) chains. These peptidomimetic molecules have a number ofparticular advantages as discussed below. For example, peptoids aresynthetic and non-natural polymers with controlled sequences andlengths, that may be made by automated solid-phase organic synthesis toinclude a wide variety of side-chains having different chemicalfunctions. Peptoids have a number of notable structural features incomparison to peptides. For example, peptoids lack amide protons; thus,no intrachain hydrogen-bond network along the polymer backbone ispossible, unless hydrogen-bond donating side-chains are put in thepeptoid chain. In addition, whereas the side-chain (“R”) groups onbiosynthetically produced peptides must be chosen from among the 20amino acids, peptoids can include a wide variety of different,non-natural side-chains because in peptoid synthesis the R group can beintroduced as a primary amine. This is in contrast to synthetic peptidesfor which the incorporation of non-natural side-chains requires the useof non-natural a-protected amino acids. Polypeptoid (or peptoids) can besynthesized in a sequence-specific fashion using an automatedsolid-phase protocol, e.g., the sub-monomer synthetic route. See, forexample, Wallace et al., Adv. Amino Acid Mimetics Peptidomimetics, 1999,2, 1-51 and references cited therein, all of which are incorporatedherein in their entirety by this reference.

The flexibility of sub-monomer synthesis allows attachment ofside-chains that satisfy the requirements of specific needs, e.g.,hydrophilicity or hydrophobicity. Another advantage of the peptoidsynthetic protocol is that it allows easy production of peptoid-peptidechimerae. In a single automated solid-phase protocol, one can alternatethe addition of peptoid monomers with the addition of α-Fmoc-protectedpeptide monomers, the latter added by standard Fmoc coupling protocolsemploying activating agents such as pyBrop or pyBop (i.e.,1H-benzotriazol-1-yloxy-tris(pyrrolidino)phosphoniumhexafluorophosphate).

Unless otherwise stated, the term “aromatic group” refers to aryl andheteroaryl groups.

The terms “substituted,” “substituted derivative” and “derivative” whenused to describe a chemical moiety means that at least one hydrogenbound to the unsubstituted chemical moiety is replaced with a differentatom or a chemical moiety. Examples of substituents include, but are notlimited to, alkyl, halogen, nitro, cyano, heterocycle, aryl, heteroaryl,amino, amide, hydroxy, ester, ether, carboxylic acid, thiol, thioester,thioether, sulfoxide, sulfone, carbamate, peptidyl, PO₃H₂, and mixturesthereof.

The term “cancer” encompasses, but is not limited to, myeloid leukemia;malignant lymphoma; lymphocytic leukemia; myeloproliferative diseases;solid tumors including benign tumors, adenocarcinomas, and sarcomas; andblood-borne tumors. The term “cancer” as used herein includes, but isnot limited to, cancers of the cervix, breast, bladder, colon, stomach,prostate, larynx, endometrium, ovary, oral cavity, kidney, testis, andlung.

The terms “compound of the present invention,” “compound of thisinvention,” “compound of the invention,” “prenylation inhibitor of thepresent invention,” “prenylation inhibitor of this invention,” and“prenylation inhibitor of the invention” are used interchangeably torefer to the compounds and complexes disclosed herein, and to theirpharmaceutically acceptable salts, solvates, hydrates, polymorphs, andclatherates thereof, and to crystalline and non-crystalline formsthereof.

The present invention is based upon the discovery that certainpyrazole-based compounds are potent prenylation inhibitors. Thesecompounds inhibit the activity of one or more of the following: GGPTaseI, GGPTase II, and FPTase. In one particular embodiment, the compoundsof the present invention, under the assay conditions disclosed in theExamples section, have an IC₅₀ value for GGPTase I of about 200 nM orless, more preferably about 60 nM or less, and most preferably about 20nM or less.

This invention is further based upon the recognition that proteinprenylation, in particular prenylation of CAAX, XXCC and/or XCXCproteins, is associated with a variety of diseases and/or conditions inplants and animals. In animals, such diseases include, but are notlimited to, cancer, restenosis, psoriasis, endometriosis,atherosclerosis, ischemia, myocardial ischemic disorders such asmyocardial infarction, high serum cholesterol levels, viral infection,fungal infections, yeast infections, bacteria and protozoa infections,proliferative disorders, and disorders related to abnormal angiogenesisincluding, but not limited to, corneal neovascularization. In plants,such diseases include yeast and viral infections.

One embodiment of the present invention provides compounds that may beused for inhibiting protein prenylation having the general structure ofFormula I:

or a pharmaceutically-acceptable salt thereof,wherein,

R₁ is a halogenated phenyl;

R₂ is nitrogen, phenyl, phenylamide, pyrazole, methylpyrazole,dimethylpyrazole, pyridine, thiophene, dimethylcyclobutyl,dimethylcyclopropyl or cyclopropyl;

R₃ is benzyl, isopropyl, chlorobenzyl, 2-benzyl-5-methyl-imidazole,methyl thiophene, trifluoro methyl benzyl, 3,5-trifluoromethyl benzyl,or ethylmethylsulfide; and,

R₄ is NH₂ or OH.

In this first embodiment, and in every other embodiment of the presentinvention, the regiochemistry of the halogenated phenyl substituent onthe pyrazole ring is shown between the two nitrogen heteroatoms toindicate that this substituent may be bound to either nitrogen atom. Thefinal composition may therefore include isolated molecules of eitherisomer or a mixture of both isomers.

Another embodiment of the present invention provides compounds that maybe used for inhibiting protein prenylation having the general structureof Formula II:

wherein

R₁ is a halogenated phenyl;

R₂ is bicyclo[2.2.1]heptane(norbornane), cyclopropane or cyclohexane;and,

R₃ is NH₂, OH or 2-ainino-3-phenylpropanamide (i.e. a compound of thestructure —NHCH(CH₂—C₆H₅)—CONH₂).

Another embodiment of the present invention provides compounds that maybe used for inhibiting protein prenylation having the general structureof Formula III:

wherein

R₁ is a halogenated phenyl;

R₂ is (CH₂)_(X) where x is an integer between 1-4, cyclobutane ordimethylcyclobutane; and,

R₃ is NH₂ or OH.

Other embodiments of the present invention provide compounds that may beused for inhibiting protein prenylation having the general structure ofFormulas IV, V or VI:

wherein R is a halogenated phenyl.

The compounds of the present invention can be synthesized from readilyavailable starting materials. Various substituents on the compounds ofthe present invention can be present in the starting compounds, added toany one of the intermediates or added after formation of the finalproducts by known methods of substitution or conversion reactions. Ifthe substituents themselves are reactive, then the substituents canthemselves be protected according to the techniques known in the art. Avariety of protecting groups are known in the art, and can be employed.Examples of many of the possible groups can be found in ProtectiveGroups in Organic Synthesis, 2nd edition, T. H. Greene and P. G. M.Wuts, John Wiley & Sons, New York, N.Y., 1991, which is incorporatedherein in its entirety by this reference. For example, nitro groups canbe added by nitration and the nitro group can be converted to othergroups, such as amino by reduction, and halogen by diazotization of theamino group and replacement of the diazo group with halogen. Acyl groupscan be added by Friedel-Crafts acylation. The acyl groups can then betransformed to the corresponding alkyl groups by various methods,including the Wolff-Kishner reduction and Clemmenson reduction. Aminogroups can be alkylated to form mono- and di-alkylamino groups; andmercapto and hydroxy groups can be alkylated to form correspondingethers. Primary alcohols can be oxidized by oxidizing agents known inthe art to form carboxylic acids or aldehydes, and secondary alcoholscan be oxidized to form ketones. Thus, substitution or alterationreactions can be employed to provide a variety of substituentsthroughout the molecule of the starting material, intermediates, or thefinal product, including isolated products.

Since the compounds of the present invention can have certainsubstituents that are necessarily present, the introduction of eachsubstituent is, of course, dependent on the specific substituentsinvolved and the chemistry necessary for their formation. Thus,consideration of how one substituent would be affected by a chemicalreaction when forming a second substituent would involve techniquesfamiliar to one of ordinary skill in the art. This would further bedependent on the ring involved.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. It is to be understood that thepresent invention encompasses any racemic, optically-active,regioisomeric or stereoisomeric form, or mixtures thereof, of a compoundof the invention, which possess the useful properties described herein,it being well known in the art how to prepare optically active forms(for example, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine prenylation inhibitor activityusing the standard tests described herein, or using other similar testswhich are well known in the art. It is also to be understood that thescope of this invention encompasses not only the various isomers whichmay exist but also the various mixtures of isomers which may be formed.For example, if the compound of the present invention contains one ormore chiral centers, the compound can be synthesized enantioselectivelyor a mixture of enantiomers and/or diastereomers can be prepared andseparated. The resolution of the compounds of the present invention,their starting materials and/or the intermediates may be carried out byknown procedures, e.g., as described in the four volume compendiumOptical Resolution Procedures for Chemical Compounds: Optical ResolutionInformation Center, Manhattan College, Riverdale, N.Y., and inEnantiomers, Racemates and Resolutions, Jean Jacques, Andre Collet andSamuel H. Wilen; John Wiley & Sons, Inc., New York, 1981, which isincorporated in its entirety by this reference. Basically, theresolution of the compounds is based on the differences in the physicalproperties of diastereomers by attachment, either chemically orenzymatically, of an enantiomerically pure moiety resulting in formsthat are separable by fractional crystallization, distillation orchromatography.

When the compound of the present invention contains an olefin moiety andsuch olefin moiety can be either cis- or trans-configuration, thecompound can be synthesized to produce cis- or trans-olefin,selectively, as the predominant product. Alternatively, the compoundcontaining an olefin moiety can be produced as a mixture of cis- andtrans-olefins and separated using known procedures, for example, bychromatography as described in W. K. Chan, et al., J. Am. Chem. Soc.,1974, 96, 3642, which is incorporated herein in its entirety by thisreference.

The compounds of the present invention form salts with acids when abasic amino function is present and salts with bases when an acidfunction, e.g., carboxylic acid or phosphonic acid, is present. All suchsalts are useful in the isolation and/or purification of the newproducts. Of particular value are the pharmaceutically acceptable saltswith both acids and bases. Suitable acids and bases are described above.In addition, hydrated, solvated and/or anhydrous forms of compoundsdisclosed herein are also encompassed in the present invention.

The compounds of present invention may be prepared by both conventionaland solid phase synthetic techniques known to those skilled in the art.Useful conventional techniques include those disclosed by U.S. Pat. Nos.5,569,769 and 5,242,940, and PCT publication No. WO 96/37476, each ofwhich are incorporated herein in their entirety by this reference.

Combinatorial synthetic techniques, however, are particularly useful forthe synthesis of the compounds of the present invention. See, e.g.,Brown, Contemporary Organic Synthesis, 1997, 216; Felder and Poppinger,Adv. Drug Res., 1997, 30, 111; Balkenhohl et al., Angew. Chem. Int. Ed.Engl., 1996, 35, 2288; Hermkens et al., Tetrahedron, 1996, 52, 4527;Hermkens et al., Tetrahedron, 1997, 53, 5643; Thompson et al., Chem.Rev., 1996, 96, 555; and Nefzi et al., Chem. Rev., 1997, 2, 449-472.

One solid phase synthetic approach useful for preparing the compounds ofthis invention is described by Marzinzik and Felder, Tetrahedron Lett.,1996, 37, 1003-1006, and Marzinzik and Felder, J. Org. Chem., 1998, 63,723-727. A general adaptation of this approach is shown in Scheme I. InScheme I, <A>, <B>, <C> and <D> represent reaction conditions suitablefor the formation of the desired products or intermediates representedby Formulas (a)-(d); W, X, Y and Z constitute

moieties within the compounds of the present invention as defined above,and R is a halogenated phenyl.

According to Scheme I, an appropriate compound is attached to a resin orother solid support under reaction conditions <A> to form a complex ofFormula (a). Appropriate reaction conditions and solid supports are wellknown to those skilled in the art. The immobilized compound of Formula(a) is then combined with a suitable reactant comprising the moieties Rand ZS to yield a compound of Formula (b). Suitable reactants for theformation of the compound of Formula (b) include, for example, ketoacids and the like, and depend upon the nature of the leaving group Land reaction conditions <B>. Suitable reactants and reaction conditionsare well known to those skilled in the art. See, e.g., March, AdvancedOrganic Chemistry 3^(rd) ed., John Wiley & Sons, Inc., New York, N.Y.,1985, pp. 435-437, which is incorporated herein by reference.

According to Scheme I, the immobilized compound of Formula (b) is thensubjected to reaction conditions <C> to form the pyrazole compound ofFormula (c), wherein R is typically as defined above, or a precursorthereto. Reaction conditions <C> are also well known to one of ordinaryskill in the art. See, e.g., March, Advanced Organic Chemistry 3^(rd)ed., John Wiley & Sons, Inc., New York, N.Y., 1985, p. 804, which isincorporated herein by this reference.

Finally, the compound of Formula (c) is cleaved from the resin underreaction conditions <D> that are well known to those skilled in the artto yield the final product of Formula (d) which, if desired, may undergopurification, crystallization or recrystallization, or further reactionsto form the compounds of this invention.

A particular embodiment of this approach is presented in Scheme II whereAA is a natural or synthetic amino acid, and X, Y, Z and R are thosedefined above.

In the first step of Scheme II, the protected amine groups bound to theresin are deprotected and reacted with a protected natural or syntheticamino acid under suitable conditions. Although both the resin-boundamine and the amino acid moiety in Scheme II are protected with Fmoc,other protecting groups well known to those skilled in the art may alsobe used. See, for example, Protective Groups in Organic Synthesis, 2ndedition, T. H. Greene and P. G. M. Wuts, John Wiley & Sons, New York,N.Y., 1991, which is incorporated in its entirety by this reference.

Removal of the amino acid protecting group and reacting the resultingfree amine with a keto acid affords the methyl ketone compound shown inScheme II. The third step of Scheme II can be carried out using any ofthe methods known to those of ordinary skill in the art of organicchemistry, including a Claisen condensation reaction. The conditionsmost suitable for this reaction may be determined using compounds suchas ethyl benzoate, such optimization may be necessary in some cases toensure that the reaction occurs without appreciable formation of sideproducts. This reaction is preferably done using dimethylacetamide (DMA)as a solvent.

The fourth step involves formation of the pyrazole ring moiety, forexample, by reacting the 1,3-diketone with an appropriately substitutedhydrazine. The final products may be cleaved from the solid-support byconventional means.

Whether or not formed using the approach of Schemes I or II, thecompounds of the present invention that are basic in nature are capableof forming a wide variety of different salts with various inorganic andorganic acids. Although such salts must be pharmaceutically acceptablein order to be administered to organisms, it may be desirable toinitially isolate compounds of the present invention from reactionmixtures as pharmaceutically unacceptable salts, which are thenconverted back to the free base compounds by treatment with an alkalinereagent, and subsequently converted to pharmaceutically acceptable acidaddition salts. The acid addition salts of the basic compounds of thisinvention are readily prepared by treating the compounds withsubstantially equivalent amounts of chosen mineral or organic acids inaqueous solvent mediums, or in suitable organic solvents such asmethanol and ethanol. Upon careful evaporation of these solvents, thedesired solid salts are readily obtained. Desired salts can also beprecipitated from solutions of the free base compounds in organicsolvents by adding to the solutions appropriate mineral or organicacids.

Those compounds of the present invention that are acidic in nature aresimilarly capable of forming base salts with various cations. As above,when a pharmaceutically acceptable salt is required, it may be desirableto initially isolate a compound of the present invention from a reactionmixture as a pharmaceutically unacceptable salt, which can then beconverted to a pharmaceutically acceptable salt in a process analogousto that described above. Examples of base salts include alkali metal oralkaline-earth metal salts and particularly sodium, amine and potassiumsalts. These salts are all prepared by conventional techniques. Thechemical bases used to prepare the pharmaceutically acceptable basesalts of this invention are those which form non-toxic base salts withthe acidic compounds of the present invention. Such non-toxic base saltsinclude those derived from pharmacologically acceptable cations such assodium, potassium, calcium, magnesium, and various amine cations. Thesesalts can easily be prepared by treating the corresponding acidiccompounds with an aqueous solution containing the desiredpharmacologically acceptable bases and then evaporating the resultingsolution to dryness, preferably under reduced pressure. They may also beprepared by mixing lower alkanolic solutions to dryness in the samemanner as before. In either case, stoichiometric quantities of reagentsare preferably employed in order to ensure completeness of reaction andmaximum yields of the desired final product.

This invention encompasses both crystalline and non-crystalline (e.g.,amorphous) forms of the salts of the compounds of this invention. Thesesalts can be used to increase the solubility or stability of thecompounds disclosed herein. They may also aid in the isolation andpurification of the compounds.

Suitable methods of synthesizing the compound of the present inventionmay yield mixtures of regioisomers and/or diastereomers. These mixtures,which are encompassed by the compounds and methods of the presentinvention, can be separated by any means known to those skilled in theart. Suitable techniques include high performance liquid chromatography(HPLC) and the formation and crystallization of chiral salts. See, e.g.,Jacques et al., Enantiomers, Racemates and Resolutions,Wiley-Interscience, New York, N.Y., 1981; Wilen et al., Tetrahedron,1977, 33, 2725; Eliel, Stereochemistry of Carbon Compounds, McGraw-Hill,New York, N.Y., 1962; and Wilen, Tables of Resolving Agents and OpticalResolutions, Eliel, ed., Univ. of Notre Dame Press, Notre Dame, Ind.,1972, p. 268. The resulting enantiomerically enriched compounds areencompassed by the present invention.

The ability of the compounds of the present invention to inhibit proteinprenylation of, for example, Ras or Ras-like proteins, may be determinedby methods known to those skilled in the art such as the methods shownin the Examples below, and by methods disclosed in the referencesincorporated herein. In certain embodiments of the present invention,compounds of the present invention are inhibitory in the GGPTase I assaydescribed in detail in Example 4. For example, compounds of the presentinvention, at a concentration of 10 μM in the GGPTase I assay describedin Example 4, preferably show a percent inhibition of at least about20%, more preferably at least about 35% and more preferably at leastabout 50%. Alternatively, compounds of the present invention, at aconcentration of 100 nM in the GGPTase I assay described in Example 4,preferably show a percent inhibition of at least about 20%, morepreferably at least about 35% and more preferably at least about 50%.GGPTase I may be prepared and purified according to the method describedby Zhang et al., J. Biol. Chem., 1994, 9, 23465-23470, and U.S. Pat. No.5,789,558, which is incorporated herein in its entirety by thisreference. GGPTase II may be prepared by a method as disclosed in, forexample, Johannes et al., Eur. J. Biochem., 1996, 239, 362-368; andWitter and Poulter, Biochemistry, 1996, 35, 10454-10463, all of whichare incorporated herein in their entirety by this reference. FPTase maybe prepared and purified by methods such as those disclosed by U.S. Pat.Nos. 5,141,851 and 5,578,477, both of which are incorporated herein intheir entirety by this reference.

The compounds of the present invention can be used for inhibitingprotein prenylation by contacting an isoprenoid transferase with thecompound. The compound can be contacted with a cell, in vitro or exvivo, and be taken up by the cell. The compounds of the presentinvention can also be administered to an organism to achieve a desiredeffect. An organism may be a plant or an animal, preferably a mammal,and more preferably a human.

For inhibiting protein prenylation in an animal, the compound of thepresent invention can be administered in a variety of forms adapted tothe chosen route of administration, i.e., orally or parenterally.Parenteral administration in this respect includes administration by thefollowing routes: intravenous; intramuscular; subcutaneous; intraocular;intrasynovial; transepithelially including transdermal, ophthalmic,sublingual and buccal; topically including ophthalmic, dermal, ocular,rectal and nasal inhalation via insufflation and aerosol;intraperitoneal; and rectal systemic.

In one particular embodiment of the present invention, proteinprenylation inhibition is used to treat or prevent conditions in anorganism due to Ras or Ras-like protein prenylation. In animals, suchdiseases include, but are not limited to, cancer, restenosis, psoriasis,endometriosis, proliferative disorders, atherosclerosis, ischemia,myocardial ischemic disorders such as myocardial infarction, high serumcholesterol levels, viral infection, fungal infections, yeast infectionsor corneal neovascularization. In plants, such diseases include yeastand viral infections.

The method of the present invention can also include the administrationof a dosage form comprising at least one compound of the presentinvention alone or in combination with other drugs or compounds. Otherdrugs or compounds that may be administered in combination with thecompounds of the present invention may aid in the treatment of thedisease or disorder being treated, or may reduce or mitigate unwantedside-effects that may result from the administration of the compounds.

The magnitude of a prophylactic or therapeutic dose of a compound of thepresent invention used in the prevention, treatment, or management of adisorder or condition can be readily determined by one of skill in theart using in vitro and in vivo assays such as those described below. Asthose of skill in the art will readily recognize, however, the magnitudeof a prophylactic or therapeutic dose of a prenylation inhibitor willvary with the severity of the disorder or condition to be treated, theroute of administration, and the specific compound used. The dose, andperhaps the dose frequency, will also vary according to the age, bodyweight, and response of the individual patient.

Typically, the physician will determine the dosage of the presenttherapeutic agents which will be most suitable for prophylaxis ortreatment and it will vary with the form of administration and theparticular compound chosen, and also, it will vary with the particularpatient under treatment. The physician will generally wish to initiatetreatment with small dosages by small increments until the optimumeffect under the circumstances is reached. The therapeutic dosage cangenerally be from about 0.1 to about 1000 mg/day, and preferably fromabout 10 to about 100 mg/day, or from about 0.1 to about 50 mg/Kg ofbody weight per day and preferably from about 0.1 to about 20 mg/Kg ofbody weight per day and can be administered in several different dosageunits. Higher dosages, on the order of about 2× to about 4×, may berequired for oral administration.

In one exemplary application, a suitable amount of a compound of thepresent invention is administered to a mammal undergoing treatment forcancer. Administration occurs in an amount of between about 0.1 mg/kgbody weight to about 20 mg/kg body weight per day, preferably betweenabout 0.5 mg/kg body weight to about 10 mg/kg body weight per day.

In another exemplary application, a suitable amount of a compound ofthis invention is administered to a mammal undergoing treatment foratherosclerosis. The magnitude of a prophylactic or therapeutic dose ofthe compound will vary with the nature and severity of the condition tobe treated, and with the particular compound and its route ofadministration. In general, however, administration of a compound of thepresent invention for treatment of atherosclerosis occurs in an amountof between about 0.1 mg/kg body weight to about 100 mg/kg of body weightper day, preferably between about 0.5 mg/kg body weight to about 10mg/kg of body weight per day.

It is recommended that children and patients aged over 65 yearsinitially receive low doses, and that they then be titrated based onindividual response(s) or blood level(s). It may be necessary to usedosages outside the ranges identified above in some cases as will beapparent to those skilled in the art. Further, it is noted that theclinician or treating physician will know how and when to adjust,interrupt, or terminate therapy in conjunction with individual patientresponse.

When used to inhibit protein prenylation in plants, the compounds of thepresent invention may be administered as aerosols using conventionalspraying techniques, or may be mixed or dissolved in the food, soiland/or water provided to the plants. Other methods of administrationknown in the art are also encompassed by the invention.

The active compound can be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or it can beenclosed in hard or soft shell gelatin capsules, or it can be compressedinto tablets, or it can be incorporated directly with the food of thediet. For oral therapeutic administration, the active compound may beincorporated with excipient and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparation can contain at leastabout 0.1% of active compound. The percentage of the compositions andpreparation can, of course, be varied and can conveniently be betweenabout 1% to about 10% of the weight of the unit. The amount of activecompound in such therapeutically useful compositions is such that asuitable dosage will be obtained. Preferred compositions or preparationsaccording to the present invention are prepared such that an oral dosageunit form contains from about 1 to about 1000 mg of active compound.

The tablets, troches, pills, capsules and the like can also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin can be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it can contain, in addition to materials of theabove type, a liquid carrier. Various other materials can be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules can be coated with shellac,sugar or both. A syrup or elixir can contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed.

The active compound can also be administered parenterally. Solutions ofthe active compound as a free base or pharmacologically acceptable saltcan be prepared in water suitably mixed with a surfactant such ashydroxypropylcellulose. Dispersion can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluidsuch that it is possible to be delivered by syringe. It can be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent of dispersion medium containing, forexample, water, ethanol, polyol (e.g., glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, e.g., sugars or sodium chloride. Prolonged absorption of theinjectable compositions may be accomplished by the inclusion of agentsdelaying absorption in the injectable preparation, e.g., aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredient into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying technique whichyield a powder of the active ingredient plus any additional desiredingredient from previously sterile-filtered solution thereof.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are employed. If desired, tablets may be coatedby standard aqueous or nonaqueous techniques.

In addition to the common dosage forms set out above, the compounds ofthe present invention may also be administered by controlled releasemeans and/or delivery devices capable of releasing the active ingredient(prenylation inhibitor) at the required rate to maintain constantpharmacological activity for a desirable period of time. Such dosageforms provide a supply of a drug to the body during a predeterminedperiod of time and thus maintain drug levels in the therapeutic rangefor longer periods of time than conventional non-controlledformulations. Examples of controlled release pharmaceutical compositionsand delivery devices that may be adapted for the administration of theactive ingredients of the present invention are described in U.S. Pat.Nos. 3,847,770; 3,916,899; 3,536,809; 3,598,123; 3,630,200; 4,008,719;4,687,610; 4,769,027; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,566; and 5,733,566, the disclosures of whichare incorporated herein in their entirety by this reference.

Pharmaceutical compositions for use in the methods of the presentinvention may be prepared by any methods known in the pharmaceuticalsciences. Such methods are well known to the art and as described, forexample, in Remington: The Science and Practice of Pharmacy, Lippincott,Williams & Wilkins, pubs, 20th edition (2000). All of these methodsinclude the step of bringing the active ingredient into association withthe carrier that constitutes one or more necessary ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelyadmixing the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product intothe desired presentation.

For example, a tablet may be prepared by compression or molding,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as powder or granules, optionallymixed with a binder, lubricant, inert diluent, surface active ordispersing agent. Molded tablets may be made by molding, in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLES Example 1

This example illustrates methods for synthesizing compounds of thepresent invention.

Coupling to Polystyrene Rink Resin

About 42 grams (g) of Fmoc-protected Rink polystyrene resin and about100 milliliter (ml) of dimethylformamide (DMF) were combined in a 500 mlpeptide vessel and shaken for about 5 minutes. The DMF was removed,about 200 ml of 20% piperidine in DMF was added to the vessel, and themixture was shaken for 30 minutes. This step was repeated prior to thesolvent being removed. Following removal of the solvent, the resin wasdeprotected by being washed 3 times with 30 ml of DMF and twice with 200ml of 1-methyl-2-pyrrolidinone (NMP). The resin was then dried for about1 hour in vacuo. About 2 equivalents of an amino acid, about 2equivalents of benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBOP), about 2 equivalents ofN-hydroxybenzotriazole (HOBt), and about 200 ml of NMP were mixed in a250 ml beaker. Before addition to the peptide vessel, containing thedeprotected Rink resin, about 4 equivalents of diisopropylethylamine(DIEA) was added to the mixture and stirred for about 1 minute. Themixture was then shaken for about 2 hours in the peptide vessel. Afterthis time, the solvent was removed and the resin was washed 3 times withabout 200 ml of NMP and 3 times with about 200 ml of dichloromethane(DCM). A ninhydrin test was performed, using standard methods, todetermine if amide formation was complete. Once the coupling wascomplete, the resin was dried overnight in vacuo.

About 5 g of the resin was suspended in about 30 ml of DMF, placed in a50 ml syringe with a polyethylene filter (available from POREXTechnologies, Fairburn, Ga.) and shaken for about 5 minutes. The DMF wasremoved, about 30 ml of 20% piperidine in DMF was added to the syringe,and the mixture was shaken for another 30 minutes. This step wasrepeated before the solvent was removed and the deprotected resin waswashed 3 times with about 30 ml of DMF and 2 times with about 30 ml ofDCM. About 1.5 equivalents of ketoacid, about 1.8 equivalents of PyBOP,about 1.8 equivalents of HOBt and about 30 ml of NMP (30 ml) werecombined. About 4 equivalents of DIEA was added to the mixture andstirred for about 1 minute. The mixture was added to the syringe andshaken for about 16 hours. After this time the solvent was removed andthe resin was washed 3 times with about 30 ml of DMF and 3 times withabout 30 ml of DCM. A ninhydrin test was used to determine if amideformation was complete. Once the coupling was complete, the resin wasdried overnight in vacuo.

About 2 g of resin complex from above was placed in a 35 ml thick-walledglass ACE pressure tube with about 10 equivalents of methyl nicotinateand about 25 ml of dimethylacetamide (DMA) and then vortexed for about 1minute. About 30 equivalents of 60% NaH in oil was added over an about30 minute period under controlled conditions; continuous vortexing, N₂blanket, periodic capping and venting. The mixture was very exothermic.The pressure tube was sealed and rotated from about 85° C. to about 90°C. for about 1 hour. The tube was allowed to cool to about 25° C. in theincubator, chilled to about 0° C., and opened behind a Plexiglas shield.The resin, with residual NaH, was slowly poured over about a 10 minuteperiod into a 500 ml peptide vessel containing about 50 ml of 15% HOAc(aq). The remaining NaH was quenched. Following the quenching, the resinwas washed with about 50 ml of 15% HOAc (aq), then 2 times with about 50ml of DMF, 2 times with about 50 ml of EtOAc, 1 time with about 50 ml ofisopropanol, 1 time with about 50 ml of MeOH, and then dried overnightin vacuo.

Library Production

About 0.05 g of each resin-bound complex set from above was dispensedinto discrete wells of a 96-well polypropylene plate (PolyfiltronicsUnifilter; 0.8 ml volume; 10 μm polypropylene filter) using a repeaterpipette and a 1:1 DMF:chloroform colloid solution of the resin (yielding48×0.25 ml aliquots). The resin was then washed 2 times with about 0.5ml of dichloromethylene and dried using a 96-well plate vacuum box.

The bottom of the 96-well plate was sealed with a TiterTop and securedto the bottom of a 96-well plate press apparatus. An about 0.7 Msolution of a selected hydrazine or substituted hydrazine in about 0.6ml of 2:1:1 DMF:mesitylene:MeOH was added to individual wells in theplate using a BioHit 8-channel pipetter. The top of the 96-well platewas sealed with another TiterTop, and the 96-well press apparatus wassealed. The plate apparatus was rotated overnight at 25° C. Followingremoval of the plate from the apparatus, the solvent was drained and theresin was washed 4 times with about 0.4 ml DMF, 2 times with about 0.4ml MeOH, 3 times with about 0.4 ml methylene chloride, and then driedfor about 1 hour using the 96-well vacuum box.

Cleavage of Product from Polystyrene Rink Resin:

About 0.4 ml of 1:1 trifluoroacetic acid (TFA):methylene chloride wasadded to each well of a semi-sealed 96-well in the 96-well plateapparatus. The 96-well plate was then shaken at 300 rpm for about 30minutes. Using the 96-well plate vacuum box, the solvent was transferredto a marked Beckman 96-well plate. The cleavage process was repeatedtwice with 1:1 TFA:DCM and the resin was washed with 1:1 acetone:DCM.The solvent in the Beckman 96-well plate was evaporated and theremaining product lyophilized 3 times with 1:1 acetonitrile:water.

¹H and ¹³C NMR spectra were obtained on a Bruker AM-250 at 250 MHz and62.9 MHz, respectively, using DMSO-d₆ as the solvent. All peaks werereferenced to the DMSO quintet at 2.49 ppm.

Molecular weight determinations were made using a PE-Sciex API 100 MSbased detector (available from Sciex, Concord, Ontario) equipped with anIon Spray Source. Flow Injection Analysis was carried out using aHTS-PAL auto sampler (available from CTC Analytics, Zwingen,Switzerland) and a HP 1100 binary pump (available from Hewlett-Packard,Palo Alto, Calif.).

The analyte was diluted to about 0.25 ml with 1:1 MeOH/CH₃CN containing1% HOAc. About 25 μL of the analyte sample was directly infused into theIon Source at about 70 μL/minutes. Electron spray ionization (ESI) massspectra was acquired in the positive ion mode. The ion-spray needle waskept at about 4500 V and the orifice and ring potentials were at about50 V and about 300V, respectively. The mass range of 150-650 Da wasscanned using a step size of 0.1 Da and a dwell time of 0.6 ms resultingin a total scan time of about 3.2 seconds.

A Gilson HPLC system consisting of two 25 ml 306 Pump Heads, a 119Variable Dual Wavelength Detector, a 215 Liquid Handler, a 811C DynamicMixer, and a 806 Manometric Module, was used for product analysis andpurification.

Analytical HPLC on the individual components of the pyrazole libraryidentified, on average, the presence of pyrazole regioisomers. Theanalytical conditions used are as follows:

Column: Thomson Instrument Co. 50×4.6 mm C18 5 μm

Flow Rate: 1 ml/minutes.

Mobile Phase A: H₂O With 0.1% Trifluoroacetic Acid (TFA)

Mobile Phase B: Methanol (CH₃OH)

Gradient: 90%-10% mobile phase A in 12-minutes.

-   -   10%-90% mobile phase B in 12-minutes.

Wavelength: 254 nm

Injection: 10 μL

Analytical HPLC conditions were optimized for Preparative HPLC of thepyrazole compounds. The preparative conditions were as follows:

Column: Thomson Instrument Co. 50×21.5 mm C18 5 μm

Flow Rate: 11 ml/minutes.

Mobile Phase A: H₂O With 0.1% Trifluoroacetic Acid (TFA)

Mobile Phase B: Methanol (CH₃OH)

Gradient: 35%-10% mobile phase A in 7 minutes.

-   -   65%-90% mobile phase B in 7 minutes.

Wavelength: 254 nm

Injection: 250 μL

N-Alkylation of Pyrazole Amides

Amide nitrogen(s) of compounds of the present invention may be alkylatedusing the following procedure:

-   1. In a scintillation vial, the pyrazole starting material (1 eq)    was dissolved in DMF.-   2. Sodium hydride (15 eq) was placed in a vial fitted with a septum    and drying tube, and the vial was shaken for 1 hour.-   3. Alkylating reagent (e.g., ethyl iodide) (15 eq) was then added,    and the reaction mixture was shaken for 5 hours.-   4. The reaction was then worked up by diluting the mixture with    ethyl acetate and washing with water and brine. The organic layers    were collected and dried over magnesium sulfate.-   5. The organic layers were filtered and concentrated in vacuo using    a Savant.-   6. Crude material was purified by flash chromatography using a    solvent system of 97:3 CH₂Cl₂:MeOH.

2-Hydroxy-4-oxo-4-pyridin-3-ylbut-2-enoic acid methyl ester (1). Acetylpyridine (1.81 mL, 16.5 mmol) and methyl oxalate (3.12 g, 26.4 mmol)were dissolved in MeOH (30 mL, anhydrous). Sodium methoxide (6.9 mL, 25%in MeOH) was added over 10 min. Reaction solidified and was completeafter 15 minutes. The solid mass was dissolved when acidified with HCl(10%, aqueous). The pH was then adjusted with NH₄OH (conc) untilprecipitation ceased. The resulting solid was taken up in EtOAc. Theaqueous layer was removed and extracted twice with EtOAc. The combinedEtOAc layers were washed with water and brine, dried over MgSO₄,filtered, and concentrated in vacuo. Collected 1 (2.90 g, 85%) as anoff-white solid.

1-(3,4-Dichlorophenyl)-5-pyridin-3-yl-1H-pyrazole-3-carboxylic acidmethyl ester hydrochloride (2). To a solution of 1 (2.90 g, 14.0 mmol)in EtOH (60 mL, anhydrous) was added 3,4-dichlorophenyl hydrazinehydrochloride (3.29 g, 15.4 mmol). The solution was heated to reflux for30 min and then cooled to 0° C. The precipitate was collected byfiltration and washed with H₂O and MeOH to yield 2 (3.79 g, 70%) as anoff-white powder.

1-(3,4-Dichlorophenyl)-5-pyridin-3-yl-1H-pyrazole-3-carboxylic acidmethyl ester hydrochloride (3). A suspension of 2 (2.0 g, 5.74 mmol) inTHF (40 mL) and H₂O (11 mL,) was treated with NaOH pellets (581 mg, 14.5mmol) and heated to reflux for 1 h. The THF was removed in vacuo and thepH of the remaining aqueous portion was adjusted to 1.5 with HCl (10%,aqueous). The resulting solid was dissolved in EtOAc. The aqueous layerwas removed and extracted with EtOAc-MeOH (4:1). The combined organiclayers were dried (brine and MgSO₄) and concentrated in vacuo to yield 3(1.61 g, 84%) as a white solid.

1-(3,4-Dichlorophenyl)-5-pyridin-3-yl-1H-pyrazole-3-carbonyl azide (4).A solution of 3 (100 mg, 0.27 mmol) and t-butyl alcohol (28.4 μL, 0.30mmol) in DMF (5 mL, anhydrous) was cooled to 0° C. Diphenylphosphorylazide (64 μL, 0.30 mmol) was added to the solution. Triethylamine (103μL, 0.60 mmol) was then added over 10 min. The solution was stirred 1 hat 0° C. and allowed to warm to room temperature and stir 16 h. Thereaction was quenched with H₂O and extracted with EtOAc. The combinedorganic layers were washed with H₂O, dried (brine and MgSO₄), filtered,and concentrated in vacuo. The resulting oil was purified by flashchromatography by eluting with hexane-EtOAc (1:1). Collected 4 (86 mg,90%) as a yellow crystalline solid.

[1-(3,4-Dichlorophenyl)-5-pyridin-3-yl-1H-pyrazol-3-yl]carbomic acidtert-butyl (5). A solution of 4 (98 mg, 0.24 mmol) and t-butyl alcohol(3 mL) were heated to reflux for 4 h. The solution was cooled andconcentrated. The resulting oil was purified by flash chromatography.Collected 4 (74 mg, 76%) as a clear oil.

1-(3,4-Dichlorophenyl)-5-pyridin-3-yl-1H-pyrazol-3-yl amine (6). TheBOC-protected compound (5, 74 mg, 0.18 mmol) was dissolved in MeOH (10mL, anhydrous) and HCl (g) was bubbled through for 10 min. The solutionwas stirred 3 h at room temperature. Concentrated in vacuo. Theremaining oil was dissolved in H₂O and neutralized with NaHCO₃ (sat.aqueous). The aqueous solution was extracted with CHCl₃; the combinedorganic layers were dried (brine and MgSO₄), filtered, and concentrated.The resulting oil was purified by flash chromatography(chloroform-MeOH—NH₄OH 95:5:0.5) to yield 6 (39 mg, 70%) as a yellowcrystalline solid.

3-endo{[1-(3,4-Dichlorophenyl)-5-pyridin-3-yl-1H-pyrazole-3-carbonyl]-amino}-bicyclo[2.2.1]heptane-2-carboxylicacid (7). A suspension of 4 (100 mg, 0.278 mmol) and3-endo-aminobicyclo[2.2.1]heptane-2-endo-carboxylic acid monohydrate(57.7 mg, 0.333 mmol) in DMF (4 mL, anhydrous) were heated to 70° C. for16 h. The solution was cooled to room temperature and diluted with H₂Oand extracted with EtOAc. The combined organic layers were dried (brineand MgSO₄), filtered, and concentrated in vacuo. The resuting oil waspurifed by flash chromatography by eluting with chloroform-MeOH—NH₄OH(80:20:0.5). Collected 7 (115 mg, 87%, R_(f) 0.16) as an oil.

1-(3,4-Dichlorophenyl)-5-pyridin-3-yl-1H-pyrazole-3-carboxylic acid[3-(1-carbamoyl-2-phenylethylcarbamoyl)-bicyclo[2.2.1]hept-2-yl]amide(8). A solution of 7 (101 mg, 0.214 mmol), HOBt (35.7 mg, 0.264 mmol),EDC (55.2 mg, 0.288 mmol), and H-Phe-NH₂ (39.5 mg, 0.252 mmol) in DMF (5ml, anhydrous) was stirred 16 h at RT. The solution was diluted with H₂Oand extracted with EtOAc. The combined organic layers were dried (brineand MgSO₄), filtered, and concentrated in vacuo. The resuting oil waspurifed by flash chromatography by eluting with chloroform-MeOH—NH₄OH(95:5:0.5). Collected 8a (67 mg, 50%, R_(f) 0.30 inchloroform-MeOH—NH₄OH (90:10:0.5)) and its diastereomer 8b (42 mg, 31%,R_(f) 0.19 in chloroform-MeOH—NH₄OH (90:10:0.5)) as crystalline solids.

Example 2

This example illustrates a method for preparing and purifying GGPTase 1.

GGPTase I was prepared and purified according to the method described byZhang et al., J. Biol. Chem., 1994, 9, 23465-23470, which isincorporated herein in its entirety by this reference.

Production of Recombinant Virus

Sf9 cells were obtained from the American Tissue Culture Collection. Thecells were maintained in Grace's medium (Gibco), supplemented with about3.3 mg/ml lactalbumin hydrolystate (Difco), about 3.3 mg/ml yeastolate(Difco), about 10% (v/v) fetal bovine serum (HyClone Laboratories,Logan, Utah), antibiotic-antimycotic mixture (Gibco), and about 0.1%Pluronic F-68 (Gibco) in 125 ml Spinner flask (available from Techne,Princeton, N.J.). To generate recombinant baculovirus, about 2×10⁶ Sf9cells were transfected with about 0.5 μg of BaculoGold wild-type viralDNA (available from PharMingen) and about 2 μg of either pVL-Fα (for αsubunit expression) or pVL-Gβ (for GGPTase-Iβ subunit expression) usingcalcium-phosphate precipitation according to the manufacturer'sinstructions (PharMingen). The virus from each transfection washarvested after about 4 days and screened using a plaque assay asdescribed by Summers and Smith, A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures, Texas AgriculturalExperimentation Station, Bulletin #1555 (1987). Recombinant virusesobtained from this screen were subjected to two further rounds of plaqueamplification to obtain purified viruses.

Production and Purification of Recombinant GGPTase-I

The purified recombinant viruses containing the cDNA sequences for the αsubunit of FPTase and GGPTase, and the β subunit of GGPTase-I were usedto co-infect about 1.5×10⁶ Sf9 cells at multiplicities of infection of5. Cells were harvested at about 65 hours post-infection bycentrifugation at about 800×g for about 15 minutes. The cells werewashed once with phosphate-buffered saline and the resulting cell pelletflash-frozen in liquid nitrogen. Cell extracts were prepared by thawingthe cell suspension in 5 volumes of about 20 mM Tris-HCl, pH 7.7, about1 mM EDTA, 1 mM EGTA, about 1 mM and a protease inhibitor mixture(Moomaw et al., Methods Enzymol., 1995, 250, 12-21), incubating the cellsuspension on ice for about one hour, and disrupting using six strokesof a Dounce homogenizer. The resulting extract was centrifuged for about1 hour at about 30,000×g, and the supernatant (designated as the solubleextract) was fractionated on a 5.0×10.0 cm column of DEAE-Sephacel(available from Pharmacia). The DEAE-Sephacel was first equilibratedwith 50 mM Tris-Cl, pH 7.7, 1 mM DTT (Buffer A) at 4° C. The solubleextract containing about 160 mg protein was loaded into the DEAE column,which was then washed with about 50 ml Buffer A and eluted with a 200 mlgradient of 0-500 mM NaCl in Buffer A. Fractions of 3 ml were collected.The fractions containing the peak of GGPTase-I activity were pooled,concentrated and exchanged into Buffer A, and then loaded into a Q-HPcolumn (1.0×20 cm, available from Pharmacia). The column was washed withabout 20 ml of buffer A and eluted with a 200 ml gradient of 0-500 mMNaCl in Buffer A. The peak fractions, containing essentially homogeneousGGPTase-I, were pooled, flash-frozen in aliquots and stored at −80° C.

Example 3

This example illustrates a method for determining GGPTase-I activity.

GGPTase-I activity was determined by the method of Casey et al., Proc.Natl. Acad. Sci. USA, 1991, 88, 8631-8635. This method measures thetransfer of isoprenoid from ³H-geranylgeranyl diphosphate (GGPP) into aRas protein with a C-terminal leucine-for-serine substitution(designated as Ras-CVLL).

Example 4

This example illustrates GGPTase I and FPTase inhibitory activities ofsome of the compounds of the present invention.

Assays for the inhibition studies of GGPTase I were performed in amanner analogous to that described by Casey, et al., Proc. Natl. Acad.Sci. USA, 1991, 88, 8631-8635, with the following modifications. Forthose assays, the reaction mixtures contained the following componentsin 50 μl:0.25 μM [³H]GGPP (sp. act. 8-10 Ci/mmol), 2.5 μM Ras-CVLL, 50mM Tris-Cl, pH 7.7, 20 mM KCl, 5 mM MgCl₂, 5 μM ZnCl₂, 1 mM DTT, 0.5 mMZwittergent 3-14 and the desired amount of the compound to be tested forinhibitory potential. After pre-equilibrating the assay mixture at 30°C. in the absence of the enzyme, the reaction was initiated by additionof the enzyme (75 ng). Following an about 10 minute incubation at about30° C., the reactions were terminated by addition of about 0.5 ml ofabout 4% SDS. About 40 mg of bovine brain membranes was added to thesamples to enhance recovery during precipitation. Product wasprecipitated by addition of about 0.5 ml of 30% TCA, allowed to stand atroom temperature for about 15 minutes, and processed by filtrationthrough glass-fiber filters as described previously (Reiss et al.,Methods: Companion to Methods in Enzymology, 1991, 1, 241-245).Reactions were never allowed to proceed to more than 10% completionbased on the limiting substrate. Assays for the inhibition studies ofFPTase were performed analogous the GGPTase I inhibition studies, except[³H]GGPP was replaced with 0.25 μM of [³H]FPP (sp. act. 8-10 ci/mmol)and Ras-CVLL was replaced with 1 μM H-Ras.

Using the method described above, the GGPTase I inhibitory activities ofsome of the compounds of the present invention were evaluated. Theresults of these tests are provided in Tables I-IV below. Theconcentration of the test compound used was 10 μM unless otherwiseindicated to be a lower concentration in parenthesis following theresult. Mixtures of regioisomers and/or enantiomers are used unlessindicated otherwise (for example, the position of substituents on acyclic or heterocyclic moiety within the backbone of the compounds ofthe present invention).

TABLE I

R1 R2 R3 R4 inhibition 3,4-di- Cyclopropane benzyl NH₂ yes chloro-phenyl 3,4-di- Pheyl (meta) benzyl NH₂ yes chloro- phenyl 3,4-di- Phenyl(para) benzyl NH₂ yes (100 nM) chloro- phenyl 3,4-di- Pyridine benzylNH₂ no (100 nM) chloro- phenyl 3,4-di- Thiophene benzyl NH₂ yes (100 nM)chloro- phenyl 3,4-di- Methyl pyrazole benzyl NH₂ no (100 nM) chloro-phenyl 3,4-di- Phenyl 2-benzyl-5-meth- NH₂ yes (100 nM) chloro- ylimidazole phenyl 3,4-di- Phenylamide 2-benzyl-5-meth- NH₂ yes (100 nM)chloro- yl imidazole phenyl 3,4-di- dimethyl pyrazole ethylmethyl NH₂yes (100 nM) chloro- sulfide phenyl 3,4-di- N Cyclohexane NH₂ yeschloro- phenyl 3,4-di- N Cyclopropane OH yes chloro- phenyl 3,4-di-Dimethylcyclobutyl Benzyl NH₂ yes (100 nM) chloro- phenyl 3,4-di-Dimethyl- Benzyl NH₂ yes (100 nM) chloro- cyclopropyl phenyl

TABLE II

R1 R2 inhibition Norbornane 2-amino-3-phenylpropanamide yes Norbornane2-amino-3-phenylpropanamide yes Norbornane 2-amino-3-phenylpropanamideyes Norbornane 2-amino-3-phenylpropanamide yes Cyclopropane2-amino-3-phenylpropanamide yes Norbornane OH yes Cyclohexane OH yes

TABLE III

R1 R2 inhibition 3,4-dichlorophenyl CH₂CH₂ yes 3-chlorophenyl CH₂CH₂ yes3,4-dichlorohenyl CH₂CH₂CH₂ yes 3-chlorophenyl CH₂CH₂CH₂ yes

TABLE IV Structure Inhibition

yes

yes

yes

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

1. A compound having a formula selected from the group consisting of:

and a pharmaceutically-acceptable salt thereof, wherein R is ahalogenated phenyl.
 2. The compound of claim 1, wherein R is3,4-dichlorophenyl.
 3. The compound of claim 1, wherein R is3-chlorophenyl.
 4. A pharmaceutical composition comprising the compoundof claim 1 and a pharmaceutically-acceptable carrier.